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Laura Asnaghi, Arushi Tripathy, Charles Eberhart; Targeting Notch signaling as a novel therapy for Retinoblastoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1662.
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© ARVO (1962-2015); The Authors (2016-present)
Retinoblastoma, a malignant tumor of the retina, is the most common intraocular cancer of childhood. Our goal was to study the role of Notch signaling in promoting growth and proliferation of retinoblastoma cells to determine if inhibiting this pathway might represent a novel approach to treatment.
WERI Rb1 and Y79 retinoblastoma lines were used as model. Expression of Notch pathway components was measured using qPCR and Western blot. Downregulation of the Jag2 ligand was achieved using short hairpin RNA (shRNA). Cell growth, proliferation, and invasion of these cell lines, upon downregulation of Jag2, were determined respectively using the colorimetric CCK8 (Cell Counting kit‑8) assay, bromodeoxyuridine incorporation and transwell invasion assays.
We found that Notch pathway components, including Jag1-2 ligands, Notch receptors and Hes1, Hey1-2 target genes, were expressed at various levels in WERI Rb1 and Y79 retinoblastoma lines. Notch ligand Jag2 RNA and protein were more highly expressed as compared to Jag1 in the retinoblastoma lines. Notch1 receptor was found to be more abundantly expressed than Notch2 and Notch3 receptors in these lines. The cleaved, active form of Notch1 was detected under standard culture conditions, further supporting a potential role for the pathway in retinoblastoma. Because Jag2 was highly expressed, we performed loss-of-function studies using shRNA. Downregulation of Jag2 significantly suppressed cell growth and proliferation by 40 to 50% in both lines, as determined respectively by CCK8 and bromodeoxyuridine assays, and partially reduced invasion, as found by transwell invasion assay.
Our findings indicate that Notch pathway components are expressed in WERI Rb1 and Y79 retinoblastoma lines, with the presence of cleaved Notch1 receptor and downstream targets further supporting pathway activity. Jag2 ligand appeared to be highly expressed and promoted retinoblastoma growth. These data suggest that Notch inhibitors represent a new potential therapeutic strategy in retinoblastoma.
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